Most of cells in human bodies consist of proteins. Most of them are generated in an inactive state and specifically activated because numerous proteases may badly affect cells. That is, the proteases control activities or lives of proteins through hydrolysis of irreversible peptide bond. Examples of such control may include assigning positions of proteins inside or outside cells or separating them from cell surfaces, activating or deactivating various growth factors, hormones, cytokines, enzymes and proteases, converting agonists into antagonists and the like.
Proteases serve to control various cell functions in a broad sense. Since such control is derived by lysing bioactive substances, the functions and roles of the proteases are very important for living of every life. For example, serious results may be caused by shortage or overexpression of a specific protease, which may result in cancer, arthritis, neurodegenerative disorder, cardiovascular disease, autoimmune disease and the like. Hence, pharmaceutical companies have concentrated on development of novel drugs based on the proteases and its matrix proteins.
Active researches are in process to investigate functions of the proteases in cells and in human bodies based on such various functions of the proteases and the recently completed genome projects. According to the human genome projects, more than 500 protease-related genes have been discovered of human genes. In recent years, it has been newly found that the proteases are pivotally associated with various human diseases, such as cancer and dementia, and also cause such diseases. For example, matrix metalloproteases (MMPs) have been recognized as factors of lysing extracellular matrix in cells and bodies. However, it has been clarified through various studies that the MMPs have a connection to an integrin signaling transfer and a cell movement responsive to lysing of pericellular matrix. It has also been discovered that the MMPs play an important role in growth of cancer, such as Angiogenesis, invasion of cancer cells, transfer of cancer cells and the like. Cell apoptosis plays a core role in a biological process based on outbreak and cure of various diseases, and also plays a pivotal role in immune systems and removal of defective cells. If the cell apoptosis does not normally is work or is damaged by pathogenic organ, the result may be fatal, and developed into incurable diseases such as cancer, Alzheimer's disease, AIDS and the like. Consequently, development of novel drugs, targeting MMPs and caspase playing a pivotal role in the cell apoptosis, are in progress around various giant pharmaceutical companies.
As new matrix proteins are found, physiological functions of various protease groups may be newly inquired, thus to be expected to find target proteins for new drugs. However, any technology for quantitatively imaging and analyzing activities and expressions of specific proteases or non-invasively imaging expression level of proteases in vivo has not been introduced. Thus, development of related techniques is urgently needed.
Several methods for detecting (measuring) proteases have been currently used. A representative thereof may include 2-D gel and multi-stage liquid chromatography, the enzyme-linked immunosorbent assay (ELISA), a method for measuring a peak shift level using a spectroscopy by conjugating (binding) a fluorophore (fluorescent substance, fluorescent material) to a peptide substrate, which is specifically lysed by proteases, or the like. However, such methods need multi-stage measurement protocols, and thus are inefficient in economical and temporal aspects in use for screening a lot of drugs, such as developing novel drugs. Furthermore, those methods are configured to detect expression of a specific protease in the body or quantitatively analyzing the expression level of the specific protease, thereby being impossible to be used for early diagnosis of diseases.
To overcome such problems, a method for detecting proteases using a molecular imaging has been developed.
The most representative technology is to use a polymer sensor for protease imaging, developed in the Harvard medical school in 2001. The sensor is configured by a chemical binding of fluorophore-peptide substrate-biocompatible polymer, which is able to be lysed by proteases. When near-infrared ray fluorophores are present close to each other within a short distance (several tens of nm), emission of the fluorophore and excitation spectra are shared. Fluorescence of the fluorophore is thusly quenched by a florescence resonance energy transfer (FRET) principle. Accordingly, when the fluorophore-peptide is connected (conjugated, bound) to polymer, fluorescence is in a quenched (non-fluorescent) state due to the physical bonding between fluorophores. Then, when the distance between fluorophores is farther due to the peptide being lysed by protease, the fluorescence is recovered such that the sensor can image an expression level of the protease. What is important in an optical imaging technology is a signal-to-noise (S/N) ratio. If high fluorescence is contained in a background, an image resolution in a target biomolecule or biologic tissue cannot help being lowered. Thus, the quenching of the fluorescence is very important. Also, if fluorescence, which has been undetected due to being quenched, is emitted from a desired biomolecule or disease portion, high S/N ratio may be exhibited, thereby obtaining a clear image. The research has exhibited the quenching of the sensor by using a self-quenching that fluorescence is quenched when near-infrared fluorophores are present close to each other. However, the self-quenching based on the FRET is performed when the fluorophores are within a very short distance therebetween and does not exhibit superior fluorescence quenching rate.
To solve the problem, various quenchers are used. Among various quenchers, a black hole quencher as a complete absorber or the like has various absorption wavelengths. As one example, BHQ-1 very efficiently absorbs fluorescence emitted at wavelengths of 480-580 nm, BHQ-2 at 550-650 nm and BHQ-3 at 620-730 nm. For a single molecule of fluorophore-peptide substrate-quencher, fluorescence is recovered when the peptide substrate is lysed by protease to allow measuring of a quantity of protease and exhibiting of a higher fluorescence quenching rate than the self-quenching of the fluorophore-fluorophore binding (conjugate, bonding). However, the fluorophore-peptide substrate-quencher may be applied only to an in vitro kit, which analyzes a quantitative amount of the protease within a mixture due to instability and cell impermeability in vivo as peptide-specific properties. In addition, the single molecule of fluorophore-peptide substrate-quencher is difficult to emit high fluorescence, capable of being imaged only in vivo, which may result in difficulty of a real-time measurement of protein expression in vivo.